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All cells have membrane proteins that form channels
to allow water and/or ions to pass through. Malfunctions have been
linked to such problems as hypertension, abnormal insulin secretion,
abnormal heart conditions and brain seizures. As a result, these
membrane proteins are often targeted by drug treatments.
Previous approaches didn't provide sufficient
resolution to let researchers accurately detect the association and
dissociation of protons to and from individual amino-acid residues in
real time.
Using the patch-clamp technique, the researchers
were able to probe the electrostatic properties of the inner lining of
the ion-channel's pore, and, from there, they inferred the rotational
angle of the pore-lining alpha-helices in the open state.
In this case, researchers focused on the muscle
nicotinic acetylcholine receptor, a membrane protein that mediates
voluntary muscle contraction.
"Our paper has implications that are specific to
this receptor, but many of the findings can be extended to several
other membrane proteins," said Claudio Grosman, a professor of
molecular and integrative physiology at Illinois.
"We are working with the open state of the ion
channel, and we now know how the helices that line the pore are
oriented. This was not known before," Grosman said. "Previous work has
told us how the helices are oriented in the closed state."
A major problem in understanding the relationship
between structure and function in proteins, and the impact that
electrostatics have on them, Grosman said, is not knowing the
protonation state of ionizable residues. Protons, he added, are so
small that they cannot be detected even with X-ray crystallographic
approaches.
For the study, Grosman and colleagues used protein
engineering. They mutated each residue of the pore's lining with basic
amino-acid residues, which can acquire a positive charge upon binding
protons and become neutral upon releasing them.
"One thing is the proton affinity of an amino-acid
residue when the amino acid is dissolved in a lot of water in, say, a
glass beaker; another thing is the affinity for protons in the complex
microenvironment presented by a membrane protein," Grosman said. "For
the first time, we were able to measure proton-transfer events at the
single-proton, single-amino-acid level, in real time. Chemists will be
happy to see this."
In addition to providing an extensive set of
proton-affinity values for basic residues, which differed greatly from
those found in a "glass beaker," the findings also discounted a
long-held theory that the rotation of the pore-lining helices underlie
the mechanism of opening and closing of the nicotinic receptor ion
channel. The data, Grosman said, indicate that such rotation is
minimal, if any. |
